DESCRIPTION (investigator's abstract): We recently used Id1 transgenes to show that not only B, but also T lymphocyte formation is dependent on the E protein family of basic helix-loop-helix transcription factors. The E proteins are also important for T cell tumor suppression. Important questions remain about when members of the Id family of transcriptional repressors are normally down-regulated and the consequences for lymphocyte development. We will now exploit recent advances in four-color flow cytometry, cell sorting and real time PCR assays to document Id expression through early stages of B and T lymphocyte differentiation. By comparing these results to a more detailed characterization of Id transgenic animals, the hypothesis will be rigorously tested that lymphoid lineage progression requires down-regulation of Id expression. Considerable progress has been made in understanding mechanisms that control Id-1 transcription and our cell line transfection studies will be extended with stromal cell-free cultures of normal bone marrow cells and EGFP reporter transgenic mice. Our findings of massive apoptosis in Id1 transgenic thymuses suggest that E proteins may function in part by regulating thresholds of responsiveness to signals delivered via TCR and cytokine receptors in lymphocyte precursors. Bone marrow and fetal thymic organ cultures will be used to more precisely define and study the apoptotic changes that we observed in transgenic mice. A potentially key observation was made that Id1 promotes differentiation of RAG-1 deficient thymocytes to the CD4+ CD8+ stage. This will be further pursued by detailed analysis of apoptosis, cell cycle status and gene expression in Id1/tg, RAG1-/- mice. Crosses between Id1 transgenic mice and Fas- or FasL-deficient animals will provide additional insight into pathways of programmed cell death that may be utilized in Id1 transgenic lymphocyte precursors. Signals mediated by cytokine and antigen receptors are likely countered by E proteins in normal situations. This will be investigated by crossing our Id transgenics with mice with altered lck activity and IL-2 receptor signaling defects to test for possible restoration of lymphocyte development. In addition to the fundamental information that will be gained about early lymphocyte development, these studies should reveal more about molecular aspects of immunodeficiency and leukemias. We expect the findings to aid the understanding of additional transcription factors that function as both regulators of cell differentiation and oncoproteins or tumor suppressors.